Mammalian spermatozoa are vulnerable to damage by peroxide reactions in the presence of O2. This damage may be the limiting factor in the lifespan of the sperm cell. Unlike the situation with regard to many other mammalian cells, almost nothing is known about the rates and mechanisms of the endogenous production of potentially harmful H2O2 and 02.- b sperm cells and of the reactions of these O2 metabolites with sperm cells. The protective enzymes which prevent cell damage in other tissues appear to be present only in part in sperm. The long range goal of this research is to fill this large gap in our knowledge, so that we may understand what governs the ultimate viability of mammalian sperm. The specific aims of this proposal are: a) To determine the rates and mechanisms of the reactions of H2O2 and O2.- with mammalian spermatozoa, and to localize the sites of these reactions; b) To determine the rates and mechanisms of the reactions leading to lipid peroxidation in mammalian sperm and to correlate these reactions with sperm viability; c) To determine the rates and source of endogenously produced H2O2 and O2.- by mammalian sperm. Sperm from rabbit (relatively H2O2-resistant) will be used in the beginning part of this project, since we have already demonstrated the existence of all these reactions in these gametes. The investigation will then be extended to mouse sperm and finally to human sperm; both are H2O2-sensitive. Highly sensitive assays for H2O2, O2.-, and lipoperoxidation, based on dual wavelength spectrophotometry and fluorimetry have been developed in this laboratory. The results anticipated from this research should provide answers to the following questions: Is it membrane damage from the lipid peroxidation that limits the ultimate lifespan of the sperm cell? Are there variations in susceptibility of damage by O2 metabolites and lipoperoxidation between sperm samples, particularly human sperm samples which might cause infertility due to abnormally short sperm lifespan? It is hoped that this new knowledge concerning O2 toxicity to spermatozoa may prrovide the explanation for cases of hitherto unexplained male infertility.